Pneumonia is a leading cause of morbidity and mortality. Understanding mechanisms of host defense in the lower respiratory tract is essential for the development of novel strategies to reduce this burden of disease. Legionella pneumophila (Lp) is an important cause of bacterial pneumonia and an intracellular pathogen of alveolar macrophages (AM). Host defense against Lp involves microbial detection by membrane-bound Toll- like receptors (TLRs), which induce pro-inflammatory cytokines, and by cytosolic nucleotide-binding leucine rich repeat proteins (NLRs), which assemble inflammasomes that activates caspase-1. Caspase-1 cleaves interleukin-1 (IL-1) and IL-18 to the active forms, favors phagosome maturation, and induces lytic cell death (pyroptosis). Recent studies have highlighted the importance of caspase-1 to the resistance of mouse bone marrow-derived macrophages (BMM) to intracellular infection with Lp, but the contribution of inflammasome activation to integrated host defense in vivo has not been studied. Furthermore, the role of caspase-1 in the resistance of human AM to Lp cannot be predicted from studies with other cell populations, as inflammasome function in human AM is uniquely muted. The overall goal of this project is to understand how key bacterial recognition events are integrated into effective immune responses in vivo, and to determine the relevance of these findings for human disease. To achieve this end we have formulated the following specific aims: Specific aim #1. Determine role of inflammasome activation in the resistance of human AM to Lp. This aim will test the hypothesis that muted function of the NLRC4 inflammasome underlies the susceptibility of human AM to parasitism by Lp. This aim will determine the ligand sensitivity of the NLRC4 inflammasome of human AM in comparison with monocytes and murine AM, determine if Lp induces or suppresses inflammasome activation in AM, and determine if stimulation of the NLRC4 inflammasome induces resistance of human AM to Lp. Specific aim #2. Determine the role of the NLRC4 inflammasome in resistance to pneumonic legionellosis. This aim will use knockout mice, a model of airborne infection, and tissue culture studies to test the hypotheses that NLR-mediated resistance to Lp vivo involves both IL-1/IL-18-dependent and -independent mechanisms that are cell-specific. Mice with lacking one or more inflammasome component and wild type mice will be exposed to aerosolized Lp and compared for bacterial clearance, cell death, and immune responses. Specific aim #3. Determine the roles of IL-1 and IL-18 in resistance to Lp pneumonia. MyD88, an adaptor that mediates signaling from TLRs and IL-1/IL-18 receptors, is required for survival from pneumonic legionellosis, but deficiencies of TLRs do not reproduce the phenotype of MyD88-/- mice. This aim will use knockout mice and blocking antibodies, a model of airborne infection, and tissue culture studies to test the hypothesis that IL-1 and IL-18 play key roles in mediating resistance to pneumonic legionellosis by stimulating interferon-? production, IL-17 responses, and directly augmenting macrophage resistance to Lp. PUBLIC HEALTH RELEVANCE: Bacterial pneumonia is a major cause of morbidity and mortality worldwide. This project addresses basic mechanisms by which bacterial pathogens are recognized within the lungs, and how these recognition pathways activate immune responses that are essential for recovery from infection. An understanding of these mechanisms will facilitate the development of novel approaches to the prevention and treatment of bacterial pneumonia.